CN115386534B - Method for culturing BHK21 cells by using serum-free culture medium and application of BHK21 cells in preparation of vaccines - Google Patents

Method for culturing BHK21 cells by using serum-free culture medium and application of BHK21 cells in preparation of vaccines Download PDF

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CN115386534B
CN115386534B CN202211314414.2A CN202211314414A CN115386534B CN 115386534 B CN115386534 B CN 115386534B CN 202211314414 A CN202211314414 A CN 202211314414A CN 115386534 B CN115386534 B CN 115386534B
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virus
bhk21 cells
chloride
serum
medium
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CN115386534A (en
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陈文庆
周劲松
徐舸辰
赵洪磊
王杰勇
类成霞
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Tianxinhe Suzhou Biotechnology Co ltd
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Abstract

The application provides a method for culturing BHK21 cells by using a serum-free culture medium and application thereof in preparing vaccines, wherein the method comprises the following steps: BHK21 cells were inoculated in DMEM/F12 medium; and culturing the BHK21 cells under conditions suitable for the proliferation of the BHK21 cells. The method can effectively reduce the culture agglomeration rate and improve the multiplication factor under the serum-free condition, improves the productivity and efficiency, and simultaneously reduces the safety risk of animal-derived additives such as serum and the like.

Description

Method for culturing BHK21 cells by using serum-free culture medium and application of BHK21 cells in preparation of vaccines
Technical Field
The invention relates to the technical field of biology, in particular to a method for culturing BHK21 cells by using a serum-free culture medium and application of the BHK21 cells in preparation of vaccines.
Background
The original cell strain of the BHK cell is a fibroblast, needs anchorage dependent growth, and can grow in suspension after being subjected to subculture. Compared with the traditional spinner flask culture, the whole process of suspension culture is closed in the cell culture process, so that intermediate open nodes are reduced, remote control can be realized, the pollution risk caused by manual operation is reduced, the volume of the culture device is increased, the difference between batches is reduced, the uniformity is improved, and the like. Due to these advantages, BHK cells cultured in suspension can be widely used for the propagation of various viral antigens and the production of veterinary vaccines. A commonly used cell is BHK21, a subcloned cell line of the BHK cell line.
However, currently, there is still a need for improvement of the culture means for BHK21 cells, particularly for BHK21 cells applied to the field of vaccines.
Disclosure of Invention
The present invention aims to solve at least one of the above technical problems to at least some extent or to at least provide a useful commercial choice. Therefore, an object of the present invention is to provide a method capable of efficiently culturing BHK21 cells using a serum-free medium.
The present application was completed based on the following findings of the inventors:
for BHK21 cells, serum was added to DMEM/F12 as a medium for virus culture. However, during the long-term development in the field of cell culture, the inventors found that there were some insurmountable defects in the suspension culture of BHK21 cells using serum as an additive in combination with DMEM/F12 medium, such as low 48-hour survival rate and high cell clumping rate of the cells, and that the components in serum inevitably elicited unwanted immune responses when applied to animals. Accordingly, the present inventors have made an effort to develop a method for culturing BHK21 cells under serum-free conditions to avoid inducing unnecessary immune responses when applied to the field of vaccines. For this purpose, the inventors have conducted extensive screening and optimization work in combination with previous rich experience accumulated in the field of cell culture, and have unexpectedly obtained a method for culturing BHK21 cells using a serum-free medium, which is capable of efficiently performing suspension culture on BHK21 cells while achieving more excellent performance than serum culture in terms of various indexes.
To this end, in a first aspect of the invention, the present application proposes a method for culturing BHK21 cells using a serum-free medium. According to an embodiment of the invention, the method comprises: BHK21 cells were inoculated in DMEM/F12 medium; and culturing the BHK21 cells under conditions suitable for the proliferation of the BHK21 cells, wherein the following components are added in advance to the DMEM/F12 medium: 0.000085 parts by weight of silver nitrate; 0.05275 parts by weight of aluminum chloride; 0.0003 part by weight of ammonium metavanadate; 0.000002 parts by weight of sodium metavanadate; 0.001015 parts by weight of cadmium chloride; 0.001315 parts by weight cobalt chloride hexahydrate; 0.00014 part by weight of chromium chloride hexahydrate; 0.014 parts by weight of copper sulfate pentahydrate; 0.00027 parts by weight of germanium dioxide; 0.00007 parts by weight of potassium bromide; 0.000085 parts by weight of potassium iodide; 0.000000124 parts by weight manganese chloride tetrahydrate; 79.5 parts by weight of magnesium chloride hexahydrate; 0.001875 parts by weight ammonium molybdate; 0.1965 parts by weight sodium metasilicate nonahydrate; 0.011 weight part of sodium selenite; 0.0023 parts by weight of sodium fluoride; 0.000065 parts by weight nickel sulfate; 0.00067 parts by weight rubidium chloride; 0.00005 parts by weight of stannous chloride; 0.0018 parts by weight of zirconium oxychloride; 0.000125 parts by weight of barium acetate; 0.001 parts by weight of lithium chloride; 2 parts by weight of recombinant human insulin; 0.03 parts by weight of hydrocortisone; 1600 parts by weight of yeast hydrolysate; 2100 parts by weight of soy hydrolysate; 1000 parts by weight of wheat hydrolysate; 3000 parts by weight of HEPES;1000 parts by weight of F68.
According to the examples of the present application, the present inventors have found that the culture conditions applicable to different cell lines are not versatile, and it is necessary to develop different culture methods based on the growth characteristics of the cells themselves so as to achieve efficient cell expansion or culture. Furthermore, the inventors have conducted a large number of optimization and screening experiments on BHK21 cell characteristics, and found that the composition of the fermentation medium significantly affects cell expansion, and the composition can not follow regularly, and the results are unpredictable, and surprisingly found that cell expansion can be achieved by adding trace elements, hormones, non-animal protein hydrolysates, buffers, and antifoaming agents using DMEM/F12 medium as the basal medium.
Further, the inventors have optimized and screened specific types of trace elements, hormones, non-animal protein hydrolysates, buffering agents and antifoaming agents, and found that silver nitrate, aluminum chloride, ammonium metavanadate, sodium metavanadate, cadmium chloride, cobalt chloride hexahydrate, chromium chloride hexahydrate, copper sulfate pentahydrate, germanium dioxide, potassium bromide, potassium iodide, manganese chloride tetrahydrate, magnesium chloride hexahydrate, ammonium molybdate, sodium metasilicate nonahydrate, sodium selenite, sodium fluoride, nickel sulfate, rubidium chloride, stannous chloride, zirconium oxychloride, barium acetate and lithium chloride are used as trace elements, recombinant human insulin and hydrocortisone are used as hormones, yeast hydrolysates, soybean hydrolysates and wheat hydrolysates are used as non-animal protein hydrolysates, HEPES is used as a buffering agent, F68 is used as an antifoaming agent, and the addition amount of each component is controlled, so that serum-free coagulation rate can be effectively reduced, the proliferation multiple can be increased, the safety risk of animal additives such as serum and the like can be reduced while the productivity and the serum-free animal protein hydrolysate and serum-free cell culture industrial application is of the present invention.
According to an embodiment of the present invention, the DMEM/F12 medium comprises:
name of raw materials Parts by weight
L-alanine 13.45
L-arginine hydrochloride 147.50
L-asparagine 22.50
L-aspartic acid 19.65
L-cysteine hydrochloride monohydrate 17.50
L-cystine dihydrochloride 31.30
L-glutamic acid 22.35
L-Glutamine 365.06
Glycine 26.75
L-histidine hydrochloride monohydrate 31.50
L-isoleucine 54.55
L-leucine 59.00
L-lysine hydrochloride 91.27
L-methionine 17.29
L-phenylalanine 35.47
L-proline 117.25
L-serine 37.25
L-threonine 53.48
L-tryptophan 19.02
L-tyrosine disodium salt anhydrous 55.30
L-valine 52.94
Phenol Red 8.10
Sodium chloride 6999.50
Monobasic sodium phosphate monohydrate 62.56
Calcium chloride dihydrate 154.50
Blue vitriod 0.001
Potassium chloride 311.80
Magnesium chloride hexahydrate 59.20
Anhydrous magnesium sulfate 48.84
Anhydrous disodium hydrogen phosphate 71.00
Zinc sulfate heptahydrate 0.43
Ferrous sulfate heptahydrate 0.42
D-Anhydrous glucose 3151.00
D-biotin 0.004
Choline chloride 8.98
D-calcium pantothenate 2.24
Folic acid 2.65
Hypoxanthine 1.95
Inositol 12.60
Linoleic acid 0.04
Lipoic acid 0.11
Nicotinamide 2.02
1,4-butanediamine dihydrochloride 0.08
Vitamin B6 0.03
Vitamin B2 0.22
Pyruvic acid sodium salt 55.00
Vitamin B1 2.17
Thymidine 0.37
Vitamin B12 0.68
Pyridoxal hydrochloride 2.00
Ferric nitrate nonahydrate 0.05
In another aspect of the invention, a method of amplifying a virus is provided. According to an embodiment of the invention, the method comprises: culturing BHK21 cells according to the method to obtain a culture solution; and inoculating the virus into the culture medium to amplify the virus. Therefore, the BHK21 cell culture solution obtained by culturing the BHK21 cells by using the serum-free culture medium according to the embodiment of the invention is beneficial to realizing virus proliferation and improving the virus titer, so that the method can be better applied to amplification production of vaccine viruses.
According to an embodiment of the invention, the virus comprises at least one of newcastle disease virus, foot and mouth disease virus and rabies virus.
In yet another aspect of the invention, a method of making a vaccine is provided. According to an embodiment of the invention, the method comprises: the virus was amplified according to the method for amplifying virus described above. Therefore, the method provided by the embodiment of the invention can be used for obtaining the vaccine with high virus titer and high yield, and the preparation method is simple and convenient to operate, high in yield and suitable for large-scale production and application.
According to an embodiment of the invention, the method further comprises: and (3) carrying out attenuation or inactivation treatment on the virus liquid obtained by amplifying the virus so as to obtain the vaccine. Therefore, adverse reaction caused by virus entering the organism can be avoided.
In yet another aspect of the invention, a vaccine is provided. According to an embodiment of the invention, the vaccine is obtained by the method for preparing a vaccine as described above. Therefore, the vaccine provided by the embodiment of the invention is safe and effective, has small adverse reaction after inoculation, and is suitable for popularization and application.
The term "vaccine" refers to an agent or composition containing an active ingredient effective to induce a therapeutic degree of immunity in a subject against a particular pathogen or disease, here therapeutically against a disease caused by an infectious virus, for example a disease caused by an infection with newcastle disease virus, foot and mouth disease virus or rabies virus, such as in particular fowl plague, rabies, foot and mouth disease, and the like.
The vaccine of the present invention may further comprise a pharmaceutically acceptable diluent, carrier, excipient and/or adjuvant. In the present context, the term "pharmaceutically acceptable" means that the diluent, carrier, excipient or adjuvant does not cause any unnecessary or adverse effect in the subject to which they are administered at the dosages and concentrations employed. The adjuvant is used for further enhancing the immune response of the body, and may be, for example, aluminum salt adjuvant, IL-12, lipopolysaccharide (LPS), etc.
The term "subject" includes, but is not limited to, humans, mice, chickens, cows, sheep, dogs, pigs, and the like.
In a further aspect of the invention, the invention provides the use of a vaccine as hereinbefore described in the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the treatment or prevention of a virus-related disease.
The vaccines of the present invention may be used to treat, alleviate or prevent a virus-associated disease or condition in a subject at risk for or suffering from such a disease or condition by stimulating an immune response in the subject by immunotherapy. Immunotherapy may comprise an initial immunization followed by additional, e.g., 1, 2, 3, or more booster immunizations. Modes of administration include, but are not limited to, intradermal, intramuscular, etc. injection, or subcutaneous, transdermal, or transmucosal administration, e.g., intranasal, oral, and the like.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
1. Preparation of culture Medium
1.1 DMEM/F12 medium
Name of raw materials mg/L
L-alanine 13.45
L-arginine hydrochloride 147.50
L-asparagine 22.50
L-aspartic acid 19.65
L-cysteine hydrochloride monohydrate 17.50
L-cystine dihydrochloride 31.30
L-glutamic acid 22.35
L-Glutamine 365.06
Glycine 26.75
L-histidine hydrochloride monohydrate 31.50
L-isoleucine 54.55
L-leucine 59.00
L-lysine hydrochloride 91.27
L-methionine 17.29
L-phenylalanine 35.47
L-proline 117.25
L-serine 37.25
L-threonine 53.48
L-tryptophan 19.02
L-tyrosine disodium salt anhydrous 55.30
L-valine 52.94
Phenol Red 8.10
Sodium chloride 6999.50
Monobasic sodium phosphate monohydrate 62.56
Calcium chloride dihydrate 154.50
Blue vitriod 0.001
Potassium chloride 311.80
Magnesium chloride hexahydrate 59.20
Anhydrous magnesium sulfate 48.84
Anhydrous disodium hydrogen phosphate 71.00
Zinc sulfate heptahydrate 0.43
Ferrous sulfate heptahydrate 0.42
D-Anhydrous glucose 3151.00
D-biotin 0.004
Choline chloride 8.98
D-calcium pantothenate 2.24
Folic acid 2.65
Hypoxanthine 1.95
Inositol 12.60
Linoleic acid 0.04
Lipoic acid 0.11
Nicotinamide 2.02
1,4-butanediamine dihydrochloride 0.08
Vitamin B6 0.03
Vitamin B2 0.22
Pyruvic acid sodium salt 55.00
Vitamin B1 2.17
Thymidine 0.37
Vitamin B12 0.68
Pyridoxal hydrochloride 2.00
Ferric nitrate nonahydrate 0.05
1.2 SFM1 medium
The following components were added to DMEM/F12 medium:
name of raw materials mg/L
Silver nitrate 0.000085
Aluminium chloride 0.05275
Ammonium metavanadate 0.0003
Sodium metavanadate 0.000002
Cadmium chloride 0.001015
Cobalt chloride hexahydrate 0.001315
Chromium chloride hexahydrate 0.00014
Blue vitriod 0.014
Germanium dioxide 0.00027
Potassium bromide 0.00007
Potassium iodide 0.000085
Manganese chloride tetrahydrate 0.000000124
Magnesium chloride hexahydrate 79.5
Ammonium molybdate 0.001875
Sodium metasilicate nonahydrate 0.1965
Sodium selenite 0.011
Sodium fluoride 0.0023
Nickel sulfate 0.000065
Rubidium chloride 0.00067
Stannous chloride 0.00005
Zirconium oxychloride 0.0018
Barium acetate 0.000125
Lithium chloride 0.001
Recombinant human insulin 2
Hydrocortisone 0.03
Yeast extract 1600
Soybean peptone 2100
Wheat peptone 1000
HEPES 3000
F68 1000
1.3 SFM2 medium
The composition differs from the SFM1 medium in that: replacing yeast extract, soybean peptone and wheat peptone with malt extract, papain soybean peptone and potato peptone respectively.
1.4 Serum culture medium
DMEM/F12 medium was supplemented with 10% serum.
1.5 Commercial culture Medium
Two commercial media were prepared according to the instructions, commercial medium 1: kinq BHK21 SFM serum-free medium, commodity medium 2: womei BHK21 SFM serum-free medium.
2. Cell culture and detection
After obtaining the SFM1, SFM2 medium, serum medium and commercial medium, BHK21 cells were cultured in suspension shake flasks in the following manner:
inoculating cells in 250ml shake flasks at 37 ℃ with 5% CO 2 And cultured in a 110rpm shaking incubator as an F1 generation for 48 hours at 0.5X 10 6 cells/ml were passaged and the culture was continued until the end of the F5 subculture. Wherein, the relative parameters of the cells are detected by a Countstar Biotech automatic cell counter at the beginning and the end of each generation of culture, and the detected cell parameters specifically comprise:
1. the initial cell density, initial cell viability, cell density, cell viability, clumping rate and cell diameter were measured at the beginning of each culture run for 48h, all of which were read by a Countstar Biotech automated cell counter.
2. Results and analysis
The results of the detection of the relevant parameters of the culture with the SFM1 medium, the SFM2 medium, the serum medium and the commercial medium are summarized as follows:
TABLE 1 results of experiments on SFM1 Medium
Figure DEST_PATH_IMAGE001
TABLE 2 results of experiments on SFM2 Medium
Figure DEST_PATH_IMAGE003
TABLE 3 serum Medium test results
Figure DEST_PATH_IMAGE005
TABLE 4 Experimental results for commercial Medium 1
Figure DEST_PATH_IMAGE007
TABLE 5 results of the experiment on commercial culture Medium 2
Figure DEST_PATH_IMAGE009
As can be seen by comparing the data in Table 1~5, the average cell density of 5.89X 10 can be achieved at 48 hours in SFM1 medium 6 cells/ml, cell proliferation is 11.8 times, compared with a serum culture medium, the cell proliferation rate is increased by 126%, and the cell agglomeration rate is reduced by 19% compared with the serum culture medium; compared with a serum culture medium, the SFM2 culture medium has the advantages that although the cell density is averagely improved by 47 percent, the cell agglomeration rate is averagely reduced by 12 percent, the cell density is still low after 48 hours (the average value is 3.73 multiplied by 10) 6 cells/ml）。
The culture effect of the SFM1 is better than that of two commercial culture mediums, compared with the commercial culture medium 1, the cell density is improved by 19.38% in 48h, and the cell agglomeration rate is reduced by 5.96%; compared with the commercial culture medium 2, the cell density is improved by 29.69% in 48h, and the cell agglomeration rate is reduced by 8.84%.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims (5)

1. A method for culturing BHK21 cells by using a serum-free medium, comprising:
BHK21 cells were inoculated in DMEM/F12 medium; and
culturing the BHK21 cells under the condition suitable for the proliferation of the BHK21 cells, wherein the following components are added into the DMEM/F12 culture medium:
0.000085mg/L silver nitrate;
0.05275mg/L of aluminum chloride;
0.0003mg/L ammonium metavanadate;
0.000002mg/L sodium metavanadate;
0.001015mg/L cadmium chloride;
0.001315mg/L cobalt chloride hexahydrate;
0.00014mg/L of chromium chloride hexahydrate;
0.014mg/L of copper sulfate pentahydrate;
0.00027mg/L germanium dioxide;
0.00007mg/L potassium bromide;
0.000085mg/L potassium iodide;
0.000000124mg/L manganese chloride tetrahydrate;
79.5mg/L magnesium chloride hexahydrate;
0.001875mg/L ammonium molybdate;
0.1965mg/L sodium metasilicate nonahydrate;
0.011mg/L sodium selenite;
0.0023mg/L sodium fluoride;
0.000065mg/L nickel sulfate;
0.00067mg/L rubidium chloride;
0.00005mg/L stannous chloride;
0.0018mg/L zirconium oxychloride;
0.000125mg/L barium acetate;
0.001mg/L of lithium chloride;
2mg/L recombinant human insulin;
0.03mg/L hydrocortisone;
1600mg/L yeast extract;
2100mg/L Soy peptone;
1000mg/L wheat peptone;
3000mg/L HEPES;
1000mg/L of F68.
2. A method for amplifying a virus, comprising:
culturing the BHK21 cells according to the method of claim 1 to obtain a culture solution; and
inoculating the virus into the culture medium to amplify the virus.
3. The method of claim 2, wherein the virus comprises at least one of newcastle disease virus, foot and mouth disease virus, and rabies virus.
4. A method of preparing a vaccine, comprising: amplifying the virus according to the method of claim 2 or 3.
5. The method of claim 4, further comprising:
and (3) carrying out attenuation or inactivation treatment on the virus liquid obtained by amplifying the virus so as to obtain the vaccine.
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